Tissue engineering is a new field that has developed with the progress of science and involves concepts and techniques from various fields of sciences, such as life science, engineering, medical science, and the like. Tissue engineering aims to understand the relationship between the structure and function of body tissues and produce a biological substitute for damaged body tissues or organs for transplantation purposes so as to maintain, improve or restore the function of human body.
A representative tissue engineering technique can be summarized as follows: removing the target tissue from a patient body; isolating cells from the removed tissue; culturing the isolated cells to allow sufficient proliferation; seeding the cells in a biodegradable porous polymer scaffold; culturing the cells in vitro for a predetermined period; and transplanting the obtained hybrid-type cell/polymer structure into the human body. After the transplantation, oxygen and nutrients are provided to the transplanted cells in the biodegradable porous polymer due to the diffusion of body fluids until blood vessels are newly formed. When the blood vessels are formed and blood is provided to the cells, the cells proliferate and differentiate, forming new tissues and organs, while the polymer scaffold is degraded and eventually disappears.
Thus, for such tissue engineering research, it is important to prepare a biodegradable porous polymer scaffold that is similar to body tissue. In order to be used as a raw material for the polymer scaffold, the material should properly serve as a matrix or frame so that tissue cells adhere to the surface of the material and form a new tissue having a three-dimensional structure. It should also be capable of serving as a middle barrier positioned between the transplanted cells and the host cells. This means that the material should be non-toxic and biocompatible such that neither blood coagulation nor an inflammatory reaction occurs after transplantation.
In addition, a polymer scaffold that is eventually completely degraded in vivo while the new tissue is being formed, such as a biodegradable polymer is an attractive candidate for a scaffolding material. Biodegradable polymers, which are currently widely being used, include polyglycolic acid (PGA), poly(L-lactic acid) (PLLA), poly(D,L-lactic acid) (PDLLA), poly(lactic-co-glycolic acid) copolymer (PLGA), poly(ε-caprolactone) (PCL), polyamino acid, polyanhydride, polyorthoester and their copolymers. However, only PGA, PLLA and PLGA have been approved by the U.S. Food & Drug Administration (FDA) as biodegradable polymers which may be used in human bodies and are used as raw materials for preparing a biodegradable porous polymer scaffold for tissue regeneration within a human body.
Various methods for preparing a porous polymer scaffold have been developed, including salt leaching, gas foaming, gas foaming salt method, fiber extrusion and fabric forming, liquid-liquid phase separation, emulsion freeze-drying, electrospinning, three-dimensional printing and the like. For example, the combination of salt leaching and phase separation involves: preparing a solvent mixture by mixing a solvent capable of dissolving a biodegradable polymer with a non-solvent which is incapable of dissolving the biodegradable polymer but is miscible with the above solvent; dissolving the biodegradable polymer in the solvent mixture; and adding an effervescent mixture to generate porosity thereto, thereby preparing a polymer mixture. The thus prepared porous biodegradable polymer scaffold has several advantages, i.e., its specific surface area and porosity are high, it is easy to regulate its pore size, it has an open pore structure, and there is no pore blockage at the surface, resulting in a more efficient introduction of stem cells into the scaffold. However, when transplanted into a living body, it is difficult to differentiate the stem cells into a specific target cell due to the absence of physiologically active substances that induce tissue regeneration.
Recently, various attempts have been made to introduce a physiologically active substance, such as growth factors or ligand peptides, into polymer scaffolds in order to improve the differentiation potential of stem cells. For instance, methods of activating stem cells by using growth factors (T. Motoki et al., Cell and Tissue Research 285: 205, 1996), methods of releasing a growth factor through gas foaming/salt leaching (J. J. Yoon et al., Biosubstances 24: 2323, 2003), and methods of stimulating the formation of bone tissue by using a porous gelatin microparticle (Z. S. Patel et al., Bone 43: 931, 2008) have been reported.
However, the above methods are problematic in that the fabrication process is relatively complicated, there is a risk of transformation of the porous biodegradable polymer scaffold taking place when its surface is modified via polymer pyrolysis, and the differentiation potential and biocompatibility are significantly reduced during the tissue regeneration of stem cells into the specific target tissue.
In order to overcome these problems, the present inventors previously developed a porous polymer scaffold with improved biocompatibility in which a peptide ligand or a growth factor is conjugated to the surface thereof (Korean Patent No. 54329). However, since in the above porous polymer scaffold, a single kind of physiologically active substance is conjugated only to the surface thereof, it is necessary for the above porous polymer scaffold to undergo in vitro cultivation with stem cells in a differentiation medium containing an additional physiologically active substance for a predetermined period, and then, transplant it in a cell/polymer construct form under partially differentiated conditions. Namely, the above porous polymer scaffold had problems in that it cannot directly induce tissue regeneration of new tissues or organs from stem cells in a living body.
Therefore, the present inventors have developed a method of efficiently inducing in situ tissue regeneration of the musculoskeletal system by using a porous biodegradable polymer scaffold in a living body, and found that when two different kinds of physiologically active substances capable of improving differentiation potential and biocompatibility are introduced into both the surface and the interior of the polymer scaffold, the thus prepared porous biodegradable polymer scaffold can efficiently induce in situ tissue regeneration of the musculoskeletal system from stem cells without in vitro cultivation with the stem cells before transplantation.